Bilirubin is a degradation of hemoglobin. It has been estimated that each day approximately 6 to 7 grams of hemoglobin is released from damaged or aged red blood cells. From this pool of hemoglobin that is rapidly destroyed within the liver, spleen, and bone marrow, approximately 200-230 milligrams of bilirubin and its derivatives are formed each day in the normal human adult. Subsequently, as a part of normal human metabolic processes the major portion of this daily bilirubin production is excreted, degraded into other derivatives, etc.
In some cases, however, an excessive amount of bilirubin occurs within the human body through overproduction of bilirubin as in the case of excessive hemolysis or by retention of bilirubin due, for example, to a liver failure. Invariably, the result of an excessive amount of bilirubin within the human body is jaundice. This widely encompassing pathological condition is characterized by markedly elevated serum bilirubin levels, for example, 10 milligrams of bilirubin per deciliter of serum or higher compared to the normal adult range of 0.1 to about 1 milligram of bilirubin per deciliter of serum, and almost always there is also present a brownish-yellow pigmentation of the skin, sclera, or mucous membranes. In addition, there is increasing evidence suggesting that excess amounts of bilirubin in the blood can lead to an undesirable increase in bilirubin concentration within body cells and interfere with various cellular processes. For example, bilirubin has been widely implicated as a potent inhibitor of many enzymatic reactions that generate energy vital to the cell. Given this background, the clinical diagnostic significance of bilirubin, in tests for liver and other related organ functions, is self-evident.
The literature on bilirubin assay methodology is quite voluminous. Good reviews outlining many of the diverse bilirubin assay techniques can be found by reference to the text entitled Clinical Chemistry-Principles and Technics, edited by R. J. Henry, D. C. Cannon, and J. W. Winkelman, Harper and Row Publishers, 2nd Edition, pages 1042-1079 (1974). A further review of bilirubin assay techniques appears in Fundamentals of Clinical Chemistry, edited by N. W. Tietz, and published by W. B. Saunders Co. pages 743-762 (1970). Perhaps the most widely used analytical procedure for bilirubin assay work has been the so-called diazo method. The diazo method employs a coupling reaction of bilirubin with a diazonium salt, such as diazosulfanilic acid, to form a pigment having an extinction coefficient higher than bilirubin by itself (which has a yellow coloration). Typically, the diazo reaction procedure for bilirubin assay includes two kinetic phases: First, a so-called "direct reaction", in which color forms quite rapidly and then an "indirect reaction", in which color develops only after the addition of methanol. As outlined in the above-noted literature reviews, particularly that of Winkelman et. al., some confusion exists in the art with respect to what these two kinetic phases actually indicate. Some people consider the direct reaction a measure of unbound or free bilirubin while the indirect reaction is considered a measure of albumin-bound bilirubin. Others have thought the direct reaction measures conjugated bilirubin while the indirect method measures the unconjugated form of bilirubin.
In addition to the above-noted confusion existing with respect to the diazo method for bilirubin assay, Winkelman et al., in their critical review of the diazo method have concluded that in view of the many variants of the diazo procedure and the complexity of the diazo reaction itself, the analytical results obtained are often different. In addition, the diazo assay method, because of its requirement of using several different reagents which must be mixed shortly before the assay determination, generally requires a fairly long period of time and can be inaccurate because of other components in human serum and other biological fluids which will also respond to diazotization.
In addition to the above-noted diazo assay method and related variants thereof for determination of bilirubin, a number of other bilirubin assay techniques have been suggested or employed at one time or another. Among others, there are various direct, spectrophotometric (i.e., colorimetric) assay techniques for bilirubin which take advantage of the molar absorbtivity inherent in bilirubin. That is, free bilirubin is a yellow pigment having a molar absorbtivity of about 5 .times. 10.sup.4 as measured at 435 nanometers. However, although the molar absorbitivity of bilirubin is high enough to be useful in various direct spectrophotometric solution techniques, it is not sufficiently high to produce a good quantitative assay of bilirubin using "dry chemistry" analytical test elements, for example, an analytical element of the type disclosed in Pryzbylowicz and Millikan, U.S. Pat. No. 3,992,158, issued Nov. 16, 1976. Thus, present-day direct spectrophotometric assay techniques for bilirubin are generally limited to solution assay techniques, particularly if accurate, quantitative results are desired. In addition, as noted in the above-referenced articles receiving various bilirubin assay techniques (see the Winkelman et al article noted above), direct spectrophotometric assay determinations for bilirubin suffer from spectral interferences due to the presence of hemoglobin which exhibits absorption peaks at 414, 540, and 576 nanometers. In addition, other materials present in bilirubin-containing biological fluids such as human serum can also cause spectral interferences using such direct spectrophotometric assay methods. For example, carotenoids can interfere with bilirubin assay because beta-carotene, one of the principal carotenoid components, exhibits an absorption peak at about 450 nm. which is in a region of the spectrum close to the absorption peak of bilirubin.
In addition to the above-noted spectral interferents for bilirubin using a direct spectrophotometric assay technique, it has been found that such techniques can also suffer interference due to the presence of other protein materials in human serum, such as albumin, to which bilirubin can bind and which as a result of such binding can cause a shift in the absorption intensity and absorption peak of bilirubin. As a result of the above-noted problems, among others, the art has had to rely substantially on the aforementioned diazo assay method for bilirubin determination or upon various modifications of the above-described direct spectrophotometric determination for bilirubin. For example, in U.S. Pat. No. 3,569,721 there is illustrated a direct spectrophotometric technique for determination of bilirubin wherein the spectral interference of hemoglobin is allegedly eliminated by, in essence, measuring the fluid sample to be tested at a wavelength for bilirubin maximum absorption and at a second wavelength for which hemoglobin, alone, is known to exhibit an absorption peak. One must then adjust the absorption peak for bilirubin concentration by an amount equivalent to the amount of hemoglobin determined to be present in the liquid sample.
Still another technique which has been used for the assay of bilirubin relates to the use of a reagent composition for bilirubin containing an organic acid or its salt such as trichloroacetic acid or an organic sulfonic acid, together with a ferric ion. In this method, bilirubin is oxidized by the organic acid or its salt in the presence of the ferric ion to a reaction product such as biliverdin and/or cholecyanin, which reaction product exhibits a characteristic blue or blue-green color and intensity which is related to the amount of bilirubin originally present. Such bilirubin assay techniques are described for example in U.S. Pat. No. 3,348,920 issued Oct. 24, 1967; U.S. Pat. No. 3,607,093 issued Sept. 21, 1971; and Belgium Pat. No. 816,927 filed Oct. 16, 1974. However, this method also suffers from many of the drawbacks noted above with respect to the diazo assay method and the direct spectrophotometric assay method. For example, the use of this assay method employing an organic acid or acid salt together with a ferric ion generally requires a substantial amount of time involving up to about 10 minutes for the reaction between the acid and bilirubin to go to completion and then an additional amount of time required to separate the final product from the original reaction media so that it may be analyzed spectrophotometrically. In addition, this test also is subject to various spectral interferents which exhibit absorption maxima in the blue region of a spectrum such as hemoglobin, various carotenoids, and the like.